ouucg



United States Patent PREPARATION OF urornornrus Edward F. Cavanaugh,Wilmette, 111., assignor to Armour and Company, Chicago, 111., acorporation of Illinois No Drawing. Application May 16, 1955, Serial No.508,825

Claims. (Ci. 260-112) Schotten' Baumann procedure which involvestreating the proteiiiaceous' substance with hydrogen peroxide prior toreaotion there'of'with'thefatty acid halide to obtain an increasedcontent of fatty acid component in the resultin'g-lipoprotein.

' In ca ooii'tained-in'the'heaction mixture to form a soap and aninorganic halide. Consequently, the lipoprotein reaction jroduct 'may becontaminated with the soap and the inunreacted alkali:

Amobject-of-this-invention is the provision of a proccas ier; preparinga purified lipoprotein. Another object is,the ,proyi si on of'a processfor separating soap and salt contaminantsr' of lipoprotein products.Other objects and, adyantages 10f thisinvention will become apparent asthe specification proceeds.

In one aspect of this invention a lipoprotein substance, obtained; byreacting a proteinaceous substance and a fatty-acid halide in analkaline mediiun and dehydrating the resulting reaction product,.-may bepurified by a process 3 which involves mixing such lipoprotein with anorganicfatsolvent having a specific gravity higher than thatof-the-lipopnotein.' The mixture of this organic fat solventand thelipoprotein substance should be achieved in a substantially anhydroussystem, and the lipoprotein substance should be maintained at an acidicreaction in such solvent' mixture. The acidification of the lipoproteinsubstance converts the soap contaminant thereof to the free fatty acid,and results in the formation of a salt therein. When this mixture isbrought to an equilibrium state, there is produced a heterogeneoussolvent system, including a salt phase subjacent to a fat solvent phase,and a lipoprotein phase superimposed upon such fat solvent phase. Then,the lipoprotein phase may be separated from the other phases in thisheterogeneous system to provide a substantially purified lipoproteinproduct.

The lipoprotein substance employed in this purification pnocedure may beprepared by reacting any proteinaceous substance With any fatty acidhalide. The term proteinaceous substance employed herein means derivedproteins such as proteoses, polypeptides and peptones, as well as wholepr0t eins.- Preferably, this fatty acid halide reactant-contains from '1to 22 carbon atoms, and espengout this Sclrotten-Baumann procedure, aportion 'ofthe fatty acid' halide reacts with the alkali:

or'ganicsalt. --Also,- suchreaction product may contain cially desirableresults are obtained with fatty acid halides containing from 12 to 18carbon atoms. Also, this fatty acid halide reactant can. be derived froma mixture of fatty acids, such as are prepared from tallow and soyabean. As suitable fatty acid halides for use in preparing thislipoprotein substance, I mention, for example, the halide derivatives ofsuch fatty acids as oleic, palmitic, lauric, capric, stearic, etc. Thehalide component of this fatty acid halide reactant may be bromine,fluorine, chlorine, etc. but better purification of the lipoproteinsubstance can be achieved when it is derived from a fatty acid chloridereactant.

The proteinaceous substance and the fatty acid halide may be reacted inan alkaline medium to form a lipoprotein reaction product. The reactionmixture should include an alkali having a cation capable of reactingwith the anion of an acid to form a salt. Better results are obtainedwhen an inorganic alklali is included in the reaction mixture, and anespecially desirable purification of the lipoprotein reaction product isachieved with an alkali having an alkali metal cation, e. g. sodium,potassium, etc.

The l-ipoprotein reaction product should be dehydrated while beingmaintained at a substantially neutral or alkaline pH. I have found thatthe acidification of such reaction product prior to drying apparentlyincreases the melting point and viscosity thereof such that it isdifficult to obtain the lipoprotein substance in a substantiallyanhydrous form. Preferably, the reaction product is maintained at a pHof at least pH 6.5 during this drying step, and especially desirabledehydration is obtained when the reaction product has a pH of at leastpH 7.5. The

drying of this lipoprotein reaction product may be ob tained by suchmethods as evaporation, lyophilization,

etc.

The dehydrated lipoprotein substance may contain such;

contaminants as soap, residual alkaliand a salt: Prepara tory topurification, the dehydrated lipdprotein substance may be pulverized'toproduce greater exposure of the contaminants therein to' the acidifyingagent and to the organic fat solvent.

minution, grinding or milling. I

out by charging the lipoprotein substance into a suitable vessel, andthen charging into the vessel the organic fat solvent. The. mixing ofthe lipoprotein substanceand the fat solvent may be achieved by pumping,stirring, churning, etc.

The acidification of the lipoprotein substance may be obtained bycontacting this solventmixture with an anhydrous acid which isdispersible in such solvent mixture. By dispersible, I mean that theanhydrous acid should be either soluble in the organic fat solvent or inthe gaseous state such that it may be bubbled through the solventmixture. I have found that substantially all of the soap contaminant ofthe lipoprotein substance may be converted to the free fatty acid byinfusing into the solvent mixture acid in such amount as to adjust thepH of the lipoprptein substance to less than about pH 5.5. I prefer toobtain conversion of the soap contaminant to the free fatty acid byadjusting the pH of the lipoprotein substance to about pH 5.0, andusually a pH within the range of pH 5.5 to 4.5 is sufiicient to obtainconversion of the soap contaminant to the free fatty acid. As acidssuitable for mixing with the solvent mixture to obtain conversion of thesoap contaminant to the free. fatty acid, I mention, for example,mineral acid anhydrides, e. g. hydrochloric, sulfuric, sulfurous andphos phoric, and organic acids, e. g. oxalic. r

Patented June 4, 1957 The pulverizing of this lipoprotein substance maybe obtained by a method such as cornthe other hand, the specific gravityof this organic fatsolvent should be less than that of the inorganicsalt. Consequently, the specific gravity of such solvent may be lessthan about 1.9 to 2.7. I mention, for example, such suitable organic fatsolvents, together with their specific gravities, as: chloroform 1.498,carbontetrachloride 1.595, trichlorethylene 1.456, dioxane 1.034,chlorobenzene 1.107, and ethylene dichloride 1.257.

The ratio of organic fat solvent to lipoprotein substance included inthis solvent mixture may be increased in accordance with an increasedconcentration of contaminant substances contained in such lipoproteinsubstance. I have found that the dehydrated lipoprotein productgenerally contains not more than 40% by weight of such contaminantsubstances, and more often the concentration of contaminants therein isless than 25% by weight. However, it is seldom that the concentration ofthese contaminants in the lipoprotein product is less than aboutbutinmore efiicient operation it may be possible to obtain aconcentration of contaminants therein of about 3 to 6%. Consequently, inthe usual practice a ratio of fat solvent to lipoprotein substance of atleast about 2 is suitable for carrying out this purification, and forpractical considerations a ratio of fat solvent to lipoprotein substanceof less than about 10 is convenient.

After dispersing the liproprotein substance in the organic fat solvent,the resulting solvent mixture may be settled to obtain an equilibriumstate therein, and to provide the heterogeneous solvent system. In thisheterogeneous system, the contaminant free fatty acid dissolves intheorganic fat solvent forming a layer subjacent to the lipoprotein phaseand superimposed upon a salt phase. The lipoprotein phase may beseparated from the other phases by draining, skimming, etc. In theformer method, the subnatant organic solvent and salt phases can bedrained from the bottom of the mixing vessel, and the residuallipoprotein recovered from the bottom of the vessel. The latterseparation method involves skimming the lipoprotein layer from the topof the mixing vessel. The residual organic fat solvent may be removedfrom the separated lipoprotein by evaporation, etc. i

This invention can be more fully illustrated by the following specificexamples:

Example I The skimmed lipoprotein layer was dried by evaporation.

This dry product was analyzed and found to contain 96% of lipoproteinand 4% of sodium chloride. The subnatant salt layer was also separatedfrom the purification mixture, and upon analysis was found to contain85% of sodium chloride.

Example II A lipoprotein product, obtained by reacting gelatin with 4 afatty acid chloride was prepared. This lipoprotein product, had a pH of53, and its composition was as follows:

Percent Lipoprotein 76.9 Sodium chloride 16.6 Free fatty acid 6.5

This lipoprotein product, in the amount of 10 gm., was suspended in cc.of trichloroethylene and the resulting mixture was settled to form threephases. The supernatant lipoprotein phase was separated by skimming, andthe skimmed lipoprotein dried by evaporation. The dry product wasanalyzed and found to have the following composition:

Percent Lipoprotein 92.6 Sodium chlor 7.4

Example III A reaction product of lard flake fatty acid chlorides andgelatin was prepared. This lipoprotein product had a pH of 7.7, and thefollowing composition:

Percent Lipoprotein 48 Free fatty acid 22.3 Sodium chlor e 25.7

This lipoprotein product, in the amount of 100 gm., was suspended in3000 cc. of ethylene dichloride. Gaseous hydrochloric acid was added tothis suspension until a pH of 4.7-5.0 was obtained. After settling, thesupernatant solvent and lipoprotein layers were separated from thesubnatant salt layer by decantation. The decanted supernatant layerswere separated by filtration. The separated lipoprotein layer was driedby evaporation, as was the subnatant inorganic salt layer. The drylipoprotein product was analyzed and found to have the following Thepurified lipoprotein product was again suspended in ethylene chloride,and the lipoprotein layer separated from the resulting-heterogeneoussystem. The separated lipoprotein layer was analyzed and found to befree from fatty acid contamination and practically free of inorganicsalt.

While in the foregoing specification various embodiments of thisinvention have been set forth and many details thereof elaborated forthe purpose of illustration, it will be apparent to those skilled in theart that this invention is susceptible to other embodiments and thatmany of these details can be varied widely without departing from thebasic concept and spirit of this invention.

I claim:

1. In a process for preparing a lipoprotein, wherein a proteinaceoussubstance and a fatty acid halide are reacted in an alkaline medium toform a lipoprotein substance, the steps of forming a substantiallyanhydrous mixture comprising said lipoprotein substance and an organicfatsolvent having a specific gravity higher than that of the lipoproteinto form a heterogeneous solvent system including a supernatantlipoprotein phase, said lipoprotein substance being at an acidicreaction in said anhydrous mixture, and separating said lipoproteinphase from other phases of said heterogeneous solvent system.

2. In a process for preparing a lipoprotein, wherein a proteinaceoussubstance and a fatty acid halide are reacted to form a lipoproteinsubstance, the. steps of forming a substantially anhydrous mixturecomprising said lipoprotein substance and an organic fat-solvent havinga specific gravity higher than that of the lipoprotein, in-

fusing said anhydrous mixture with an anhydrous acidformed therein inassociation with said lipoprotein, settling said mixture to form aheterogeneous solvent system including a supernatant lipoprotein phase,and separating said lipoprotein phase from other phases of saidheterogeneous solvent system.

3. The process of claim 2 in which said fatty acid halide is an oleicacid halide.

4. The process of claim 2 in which said fatty acid halide is a fattyacid chloride.

5. The process of claim 2 in which said proteinaceous substance isgelatin.

6. In a process for preparing a lipoprotein, wherein a proteinaceoussubstance and a fatty acid halide are reacted in an aqueous mediumcontaining an alkali having an alkali metal cation component to form alipoprotein substance, the steps of dehydrating said lipoproteinsubstance, mixing the dehydrated lipoprotein substance with an organicfat solvent having a specific gravity higher than that of thelipoprotein, acidifying the resulting mixture with an anhydrous gaseousmineral acid to form a substantially anhydrous heterogeneous solventsystem including a supernatant lipoprotein phase, and separating saidlipoprotein phase from other phases in said heterogeneous solventsystem.

7. The process of claim 6 in which said alkali metal 25 cation componentis a sodium component.

8. The process of claim 6 in which said anhydrous gaseous mineral acidis gaseous hydrochloric acid.

9. In a process for preparing a lipoprotein, wherein a 6 proteinaceoussubstance and a fatty acid chloride are reacted in an aqueous mediumcontaining an alkalinizing agent having an alkali metal cation componentand wherein the resulting condensation product is substantiallydehydrated while being maintained at a pH of at least 6.5, the steps ofmixing said dehydrated condensation product with an organic fat solventhaving a specific gravity higher than that of said lipoprotein, infusinginto the resulting solvent mixture an anhydrous organicsolvent-dispersible acid in such amount as to adjust the pH of saidcondensation product to within the range of pH 4.5 to 5.5, andseparating the supernatant lipoprotein phase from the other phases ofsaid solvent mixture.

10. The process of claim 9 in which said organic fat solvent is ethylenedichloride.

References Cited in the file of this patent UNITED STATES PATENTS2,113,819 Tucker Apr. 12, 1938 2,454,915 Fevold et a1. Nov. 30, 19482,460,980 Fraenkel-courat et al. Feb. 8, 1949 2,603,630 Aries July 15,1952 OTHER REFERENCES MacArdle: Use of Solvents (Van Nostrand) pp. 129-130, 133-134 (1925).

Anson et 21.: Advances in Protein Chem, vol. 1, page 20 (1944).

1. IN A PROCESS FOR PREPARING A LIPOPROTEIN, WHEREIN A PROTEINACEOUSSUBSTANCE AND A FATTY AID HALIDE ARE REACTED IN AN ALKALINE MEDIUM TOFORM A LIPOPROTEIN SUBSTANCE, THE STEPS OF FORMING A SUBSTANTIALLYANHYDROUS MIXTURE COMPRISING SAID LIPOPROTEIN SUBSTANCE AND AN ORGANICFAT SOLVENT HAVING A SPECIFIC GRAVITY HIGHER THAN THAT OF THELIPOPROTEIN TO FORM A HETEROGENEOUS SOLVENT SYSTEM INCLUDING ASUPERNATANT LIPOPROTEIN PHASE, SAID LIPOPROTEIN SUBSTANCE BEING AT ANACIDIC REACTION IN SAID ANHYDROUS MIXTURE, AND SEPARATING SAIDLIPOPROTEIN PHASE FROM OTHER PHASES OF SAID HETEROGENEOUS SOLVENTSYSTEM.